The Signal Transduction Mechanism Of Exogenous EGF On The Proliferation Of FL Cells And The Study Of Its Safety, Exploitation Of Medicinal Herbs Resource

Epidermal growth factor(EGF),a powerful cell division factor,was originally secretion from salivary gland cells.EGF has promotion effect on epithelial cells. Preliminary studies in humans suggest that exogenous EGF enhances healing of skin and cornea wounds or ulcers.The concentration of EGF is higher in gastrointestinal tract than in blood circulation.Recently,the study of exogenous EGF in treating the benign diseases of gastrointestinal tract becomes a hotspot in this area owing to the protection effect of EGF on alimentary canal.Some researches also find that EGF and the antagonist of EGFR can contribute to the occurrence,development or decrescence of the gastrointestinal tumors,therefore the application of EGF on clinic is cautious.The signal transduction mechanism of EGF on the proliferation and the activation of EGFR are very complex.Abnormal expression of Bcl-2,p53 concemed with occurrence and development of tumor,so lots of researches are need to illustrate the mechanism of its clinical effects.We first analysis the proliferation of FL cells stimulated by EGF of various concentration and time,then detect the effect of EGF on Ras/Raf/MEK/ERK signaling transduction pathway(s)and the change of Bcl-2 and p53 protein,provide theory and experimental data for the application of EGF on entirety animal experiment.Now,the main source of EGF products applied on clinical come from gene recombinant or isolation from the submaxillary glands of mices.But their disadvantages,such as high investment,shortness of source etc,are hardly to be overcomed,so it is difficult to utilize EGF on clinic extensively.If we can't solve this problem,even our study has illuminated the signal transduction mechanism and demonstrated the effect of EGF in treating the disease,it will also be valueless.If we can extract inexpensive bioactive EGF from the submaxillary glands of goats expediently,it will benefit the nation and the people overwhelmingly.Being a fundamental economic animal,native material of goats was abundance and the cost is cheap,all of these provide a spacious perspective.There are two parts of this thesis.Partâ… :The signal transduction mechanism of exogenous EGF on the proliferation of human amnion cells and the study of its safetyObjectionTo explore the effect of EGF on Ras/Raf/MEK/ERK signal transduction pathway(s) and the change of Bcl-2 and p53 protein when FL cells were promoted by rhEGFMaterial and methods1.The proliferation of FL cells was evaluated by MTT assay:FL cells were cultured in MEM containing 10%FBS,incubated in 37℃,5%CO₂ for 48 hours,colleted the cells,wash with PBS solution,dilluted to 2×10⁸ cell /L number,then added 100 ul samples to every reaction.When cells stuck wall,cultured them in serum-free medium containing rhEGF of various concentration(0.1,1,10,30, 60μg/L)for 1,2,3,4,5 days,or containing rhEGF(10μg/L)and PD98059 of various concentration(25,50,75μmol/L)for 48 hours.In control group,rhEGF was substituted with saline solution.Then added MTT(5 mg/ml)20ul and cultured for another 4 hours, added DMSO 150ul to stop the reaction.Read the absorbance value on OD490 nm,the cell growth acceleration rate was calculated as follows acceleration rate=(Ai-Ao/(Ao)×100% Ao,Ai:absorbance value of control group and experiment group respectively.2.Western blot was used to detect Ras/Raf/MEK/ERK signaling transduction pathway(s)and the expression of Bcl-2 and p53 protein:When cells stuck wall,cultured in serum-free medium containing rhEGF of various concentration(1,10,60μg/L).In control group,rhEGF was substituted with saline solution.37℃,5%CO₂ incubated for 5 min - 4 h,colleted the cells,added cell disruption lysate,collected supernate and detected the concentration of protein,added 50μg samples to every reaction.After electrophoresis,transfer protein to PVDF membrane,blocked with 5%BSA in 4℃for 2 hours.Added first antibody,enzyme labeled second antibody reaction for 2 hours or overnight,washed with PBST for 3 times, added ECL reagent for 1.5 min,then exposed to theâ…©film.3.To explore the effect of MAPK pathway inhibitor PD98059 on the proliferation of FL cell and the activation of ERK1/2:FL cells were cultured with PD98059 of various concentration(25,50,75μmol/L) for 1 h,then added 10μg/L rhEGF,cultured for 30 min,Western blot was used to detect the change of ERK1/2 protein.Results1.Incubated in 37℃,5%CO₂ for 48 hours,Morphology of FL cells cultured in MEM medium with rhEGF became fusiform mostly.2.At 1st day to 5th day,rhEGF of various concentration(1,10,30,60μg/L)can significantly accelerate the growth of FL cells and there was no significant difference when rhEGF concentration was 0.1μg/L and.The proliferation rate of FL cells reached the maximum on third day when concentration of EGF were 10μg/L(42.4%,P＜0.01) and 60μg/L(36.1%,P＜0.01)respectively.At 4th day to 5th day,the proliferation rate of FL cells began to decline.3.Stimulated by rhEGF of various concentration for 30 min,Western blotting showed that the activity of p-Raf increased significantly when rhEGF ranged in 1μg/L～60μg/L(P＜0.05).Furthermore,we detect the level of p-Raf when FL cells were treated with 10μg/L rhEGF of different time(0 min,5 min,30 min,1 h,2 h respectively).Result showed the expression of p-Raf protein was highest at 5 min(P＜0.01),then began to decline.2 h after cells were treated with rhEGF,the level of p-Raf had no significant difference when compared with control group(P＞0.05).4.Stimulated by rhEGF of various concentration for 30 min,Western blot showed that the activity of p-ERK1/2 increased significantly when rhEGF ranged in 1μg/L～60μg/L(P＜0.01).Further more,we detect the level of p-ERK1/2 when FL cells were treated with 10μg/L rhEGF of different time(0 min,5 min,30 min,1 h,2 h respectively).Result showed the expression of p-ERK1/2 protein was highest at 5 min(P ＜0.01),then began to decline.2 h after cells were treated with rhEGF,the level of p-ERK1/2 was lower when compared with control group(P＜0.05).5.Compared with control group,Western blot showed PD98059 of various concentration(25,50,75μmol/L)can inhibit the expressive of p-ERK1/2.The effect of PD98059 has dose dependent.6.Stimulated by rhEGF of various concentration for 1 hour,Western blot showed that the activity of Bcl-2 protein increased significantly when rhEGF ranged in 1μg/L～60μg/L(P＜0.05),but p53 protein decreased significantly.The effect reach maximal when rhEGF in 10μg/L but 1μg/L rhEGF was minimum,there was significant difference between two groups(P＜0.01).7.We detect the level of Bcl-2 when FL cells were treated with 10μg/L rhEGF of different time.Result showed the expression of Bcl-2 protein was highest at 1 h(P＜0.01),then began to descend.It is similar with the change of p-ERK1/2 when compared with control group.ConclusionsrhEGF stimulated FL cell proliferation,Ras/Raf/MEK/ERK signaling transduction pathway in a time or dose dependent manner.EGF of 10μg/L concentration had the best effect.FL cells stimulated by rhEGF had self-adapting control action.EGF activate Bcl-2 and p53 also in a time or dose dependent manner.It was saft when rhEGF concentration was in 1μg/L.Partâ…¡:The isolation,purification and identification of EGF from goats' submaxillary glandsObjectionIsolate,purify and identify EGF from goats' submaxillary glandsMaterial and methods1.Isolation and purification EGF from the goats' submaxillary glands:We took several fresh or freezed goats' submaxillary glands,cut off connective tissue and smashed it with blender,then put it into homogenizer.Added HAC(4℃),centrifigated 50 000G for 30 mins,collected the supernatant.Pass it through DEAE Sephadacel anion exchange column,collected the eluent,and detected the protein flow peak at wave length 280 nm.ELISA test was done to detect active component of EGF,designed 0.2 M ammonium acetate as negative control,EGF standard preparation as positive control. Read the number on OD652 nm or on OD490 nm(added 50 ul 2 M sulphuric acid to stop the reaction).Component whose reading are close to EGF standard preparation were considered active constituent.Undertook dialyze to take off saline matter from active constituent,molecular weight of bag filter was 3500,then condense with glycerin or PEG(molecular weight:20 000)to remove water.All the proceed was on ice(4℃). ELISA test was done to detect active component of EGF of concentrated solution,then took the concentrated solution pass through Sephadex G50 column.Detected the protein flow peak at wave length 280nm.Collected the eluent,and ELISA test was done to detect active component of EGF.Consequence,the absorbance of one protein flow peak was higher than others.Therefore,this protein flow peak was the peak of EGF identified by the ELISA.2.Identification the bioactivity of goats' EGF2.1.FL cells were stimulated by goats' EGF of various concentration.The proliferation of FL cells were evaluated by MTT assay.2.2.FL cells were stimulated by goats' EGF of various concentration.The cell cycle was detected by flow cytometry.2.3.SDS-PAGE electrophoresis was used to detect goats'EGF band.Results1.Incubated in 37℃,5%CO₂ for 48 hours,morphology of FL cells cultured in MEM medium with rhEGF became fusiform mostly.2.MTT methods:MTT methods showed the cell growth acceleration rate of goat EGF group(diluted 1∶100,1∶500,1∶1000 respectively)and rhEGF group were 20.9%,39.4%,24.9%and 40.6%respectively.So the goats' EGF can significantly accelerate the growth of FL cells(P＜0.01).The effect of goats' EGF diluted to 1∶500 was best and there was no significant difference with the rhEGF(P＞0.05).3.Flow cytometry:compare with control group,goats' EGF also increased the S-phase cells fraction obviously,G₀/G₁ phase cells fraction decreased from 71.1±1.9 to 60.3±2.2(P＜0.01),S-phase cells fraction increased from 18.8±1.4 to 25.8±1.1(P＜0.01).4.SDS-PAGE electrophoresis showed a band in 6 KD.ConclusionsThe protein isolated from goats' submaxillary glands was identified as EGF,it accelerates the growth of FL cells,changes the morphology of the cultured cells and increases the S-phase cells fraction obviously.Therefore it is bioactive EGF.Goats' submaxillary glands can be the medicinal herbs resource of EGF.